Methods for the identification of peptidyl compounds interacting with extracellular target molecules

Abstract

The present invention provides libraries expressing peptide libraries on the extracellular cell surface of host cells and methods for identifying peptides that bind extracellular target molecules under the physiological conditions encountered in biological fluids and secretions. The present invention is also directed to vectors for expressing gene fusion proteins and for targeting those fusion proteins to the extracellular cell surface.

1 . A method for identifying peptides that specifically bind to extracellular target molecules, comprising: 
 introducing an expression library comprising a plurality of oligonucleotides, at least a majority of the oligonucleotides having different sequences encoding different peptides, into a first plurality of mammalian host cells, the host cells expressing and displaying the peptides on an extracellular cell surface;    contacting the host cells displaying the peptides with at least one extracellular target molecule under substantially physiological conditions;    selecting from the first plurality of host cells displaying the peptides a first subset of cells displaying peptides that bind to the at least one target molecule, and    recovering from the first subset of host cells a first sub-library of the expression library comprising at least one oligonucleotide that encodes a peptide that binds to the at least one target molecule.    
     
     
         2 . The method of  claim 1 , wherein the contacting is performed in the presence of a complex biological fluid.  
     
     
         3 . The method of  claim 2 , wherein the complex biological fluid is blood, serum, plasma, sweat, tears, urine, semen, vaginal fluid or mucous.  
     
     
         4 - 6 . (canceled)  
     
     
         7 . The method of  claim 1 , further comprising: 
 introducing the first sub-library into a second plurality of host cells, the second plurality of host cells expressing peptides encoded by the first sub-library and displaying the peptides on the extracellular cell surface;    contacting the second plurality of host cells displaying the peptides with the at least one extracellular target molecule;    selecting from the second plurality of host cells a second subset of host cells displaying peptides that bind to the at least one target molecule; and    recovering from the second subset of selected host cells a second sub-library comprising at least one oligonucleotide that encodes the peptide that binds to the at least one target molecule.    
     
     
         8 . (canceled)  
     
     
         9 . The method of  claim 1 , wherein the target molecule is an extracellular protein, a carbohydrate, or a lipid.  
     
     
         10 . The method of  claim 9 , wherein the extracellular protein is a peptide, protein, antibody, glycoprotein, phosphoprotein, glycophosphoprotein, proteoglycan, or a polymeric complex thereof.  
     
     
         11 - 12 . (canceled)  
     
     
         13 . The method of  claim 1 , wherein the target molecule is displayed on an extracellular surface of a target cell.  
     
     
         14 . The method of  claim 13 , wherein binding of the peptide to the target molecule results in a change in a detectable phenotype of the target cell.  
     
     
         15 . The method of  claim 14 , wherein the change in detectable phenotype is 
 (a) a change resulting from altering a function of a mutant protein; (b) an alleviation of factor-dependent growth; or (c) a change in apoptotic state of the target cell.    
     
     
         16 - 17 . (canceled)  
     
     
         18 . The method of  claim 1 , wherein the target molecule is displayed on an extracellular surface of an animal cell.  
     
     
         19 . The method of  claim 18 , wherein the animal cell is a mammalian cell.  
     
     
         20 . The method of  claim 18 , wherein the selecting comprises 
 contacting the animal cell with a detectably labeled antibody that binds a marker on the animal cell and detecting the bound, labeled antibody.    
     
     
         21 . The method of  claim 18 , wherein the selecting comprises contacting the host cells with a detectably labeled antibody that binds a marker on the host cells and detecting the bound, labeled antibody.  
     
     
         22 - 23 . (canceled)  
     
     
         24 . The method of  claim 1 , wherein the target molecule is (a) a molecule that binds to a cell surface receptor; (b) an antibody, wherein the binding of the peptide to the antibody reduces binding of the antibody to an antigen; (c) an extracellular enzyme, wherein the binding of the peptide to the enzyme alters the activity of the enzyme; or (d) a tumor-specific antigen or a tumor-associated antigen.  
     
     
         25 . The method of  claim 24 , wherein the target molecule is a cytokine, a chemokine, or a secreted factor; and the binding of the peptide to the target molecule, alters binding of the target molecule to the cell surface receptor.  
     
     
         26 . The method of  claim 25 , wherein the binding of the target molecule to the cell surface receptor is reduced.  
     
     
         27 . (canceled)  
     
     
         28 . The method of  claim 24 , wherein the antibody is an autoantibody.  
     
     
         29 . (canceled)  
     
     
         30 . The method of  claim 24 , wherein (i) the peptide binds to an active site of the extracellular enzyme, thereby inhibiting activity of the enzyme; or (ii) the peptide binds to an allosteric regulatory site on the extracellular enzyme.  
     
     
         31 . The method of  claim 24 , wherein the peptide is a competitive inhibitor of substrate binding to the extracellular enzyme.  
     
     
         32 - 33 . (canceled)  
     
     
         34 . The method of  claim 1 , wherein the target molecule is displayed on an extracellular surface of a target cell.  
     
     
         35 . The method of  claim 34 , wherein the target molecule is a mutant protein, and binding of the peptide alters a function of the mutant protein.  
     
     
         36 . The method of  claim 34 , wherein peptide binding alleviates factor-dependent growth; changes an apoptotic state of the target cell; or causes increased or decreased sensitivity to a cytotoxic drug.  
     
     
         37 - 38 . (canceled)  
     
     
         39 . The method of  claim 1 , wherein the peptides are displayed as a fusion protein with a presentation molecule.  
     
     
         40 . The method of  claim 39 , wherein the presentation molecule is CD24 or IL-3.  
     
     
         41 . (canceled)  
     
     
         42 . The method of  claim 39 , wherein the fusion protein further comprises an epitope.  
     
     
         43 . The method of  claim 42 , wherein the epitope is polyhistidine, V5, FLAG, or myc.  
     
     
         44 . The method of  claim 39 , wherein the fusion protein further comprises a signal for a glycophosphatidylinositol anchorage.  
     
     
         45 . The method of  claim 1 , wherein the selecting comprises use of a flow sorter to identify the first subset of cells exhibiting binding to the target molecule.  
     
     
         46 . The method of  claim 34 , wherein the target cells are coupled to magnetic beads and the selecting comprises collection of the first subset of cell bound to the target cells.  
     
     
         47 . The method of  claim 1 , wherein the target molecule is detectably labeled and the selecting comprises identifying host cells bound to the labeled target molecule.  
     
     
         48 . The method of  claim 1 , wherein the target molecule comprises detectably labeled antibody and the selecting comprises identifying host cells bound to the labeled antibodies.  
     
     
         49 . The method of  claim 1 , wherein the target molecule is a protein associated with an autosomal dominant disease, with an oncogenic disease, or with normal cellular function.  
     
     
         50 . A peptide display library, comprising: 
 a plurality of at least one type of expression vector, each expression vector having    a first nucleic acid sequence encoding a signal sequence, a presentation molecule comprising modified CD24 or modified IL-3 receptor, and a transmembrane domain, and    a cloning site for insertion of a second nucleic acid sequence distal to the transmembrane domain, the second nucleic acid sequence encoding an amino acid sequence;    whereby fusion proteins are expressed and displayed on an extracellular surface of a host cell.    
     
     
         51 . The library of  claim 50 , wherein the second nucleic acid sequence encodes peptides having up to 20 amino acids.  
     
     
         52 . A library peptide displayed as an extracellular membrane protein fusion, comprising: 
 modified CD24 comprising a signal sequence, a library peptide inserted within CD24 amino acid sequence, and a transmembrane domain;    whereby the peptide is displayed on an extracellular cell surface such that the peptide can interact with extracellular target molecules under substantially physiological conditions.    
     
     
         53 . A plasmid expression vector, comprising: 
 an SV40 origin of replication; and    a nucleic acid sequence comprising SV40 early promoter region and SV40 Large T antigen coding region, the SV40 early region promoter promoting transcription of the Large T antigen coding region;    whereby replication of the plasmid is self-regulating.    
     
     
         54 - 68 . (canceled)  
     
     
         69 . The method of  claim 2 , wherein the complex biological fluid is substantially undiluted.